RU2798396C2 - Composition or a method comprising (t)ew-7197 for the treatment or prevention of corneal endothelial diseases - Google Patents

Abstract

FIELD: medicine, ophthalmology.

SUBSTANCE: invention is intended for the treatment or prevention of a condition, disorder or disease of the corneal endothelium. Compound (T)EW-7197(N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof is used either for treatment or prevention of a condition, disorder or disease of the corneal endothelium.

EFFECT: effective treatment or prevention of a condition, disorder or disease of the corneal endothelium.

10 cl, 9 dwg, 1 tbl, 9 ex

Description

[Technical field]

[0001]

The present invention relates to a novel use of (T)EW-7197. More specifically, the present invention relates to technology, method, or agents for treating or preventing a condition, disorder, or disease of the corneal endothelium, comprising (T)EW-7197 (N-((4-([1,2,4]triazolo[1, 5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or solvate, and technologies for the conservation of corneal endothelial cells using this technology.

[Prior Art]

[0002]

Visual information is recognized when light transmitted to the cornea, which is the transparent tissue at the front of the eyeball, reaches the retina and excites the nerve cells in the retina, and the generated electrical signal is transmitted through the optic nerve to the visual cortex of the brain. To achieve good vision, the cornea must be transparent. The transparency of the cornea is maintained by maintaining a constant water content with the help of a pump and by the protective functions of the corneal endothelial cells.

[0003]

While human corneal endothelial cells are present at a density of approximately 3000 cells/mm ² at birth, the ability to regenerate after injury is very limited. One of the endothelial diseases of the cornea, Fuchs endothelial corneal dystrophy, is a disease that causes an abnormality of endothelial cells within the cornea, resulting in corneal edema. The reason for this is unknown. In Fuchs endothelial corneal dystrophy, an extracellular matrix such as collagen is deposited on part of the posterior surface of the Descemet's membrane in the posterior part of the cornea, resulting in droplet-like endothelial corneal changes and thickening of the Descemet's membrane. Endothelial corneal changes in the form of drops and thickening of the Descemet's membrane are the causes of glare or blurred vision in patients with Fuchs endothelial corneal dystrophy, which compromises the quality of life of patients. It is clear that there is no effective therapeutic method other than corneal transplantation for Fuchs endothelial corneal dystrophy. However, there is a shortage of corneal donation in Japan, where the number of patients waiting for corneal transplantation is about 2600, while the number of corneal transplants performed in Japan is about 1700 annually.

[Summary of the present invention]

[Solution to the problem]

[0004]

The inventors found that (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl )-1H-imidazol-2-yl)methyl)-2-fluoroaniline) with the following structure:

[Chemical formula 1]

exhibits a very good effect of suppressing cellular disorders in the cells of the corneal endothelial disorder model. The effect of suppressing cellular disorders (T) of EW-7197 was confirmed even at a very low concentration. In addition, the inventors have also found that (T)EW-7197 can suppress the expression of an extracellular matrix (ECM) such as fibronectin for the treatment of ECM disorders in corneal endothelial disorders in corneal endothelial disorder model cells.

[0005]

Therefore, the present invention relates, for example, to the following items.

(Paragraph 1)

A composition for treating or preventing a condition, disorder or disease of the corneal endothelium, comprising (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)- 5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof or a pharmaceutically acceptable salt or solvate thereof.

(Point 2)

The composition according to claim 1, wherein the composition is intended to suppress the decrease in the density of corneal endothelial cells.

(Point 3)

The composition according to claim 1 or 2, wherein the condition, disorder or disease of the corneal endothelium is Fuchs endothelial corneal dystrophy or endothelial corneal changes in the form of drops.

(Item 4)

The composition of claim 1, wherein the condition, disorder or disease of the corneal endothelium results from overexpression of the extracellular matrix (ECM).

(Item 5)

The composition of claim 4, wherein the extracellular matrix (ECM) is selected from the group consisting of type I collagen, type IV collagen, type V collagen, and fibronectin.

(Item 6)

The composition according to claim 4 or 5, wherein the condition, disorder or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, droplet formation, thickening of the Descemet's membrane, thickening of the cornea, corneal opacity, scar, corneal opacity, corneal macula, leukoma, glare, and defocused vision.

(Item 7)

The pharmaceutical composition of any one of 1 to 6 wherein the condition, disorder or disease of the corneal endothelium is Fuchs' corneal endothelial dystrophy.

(Item 8)

A composition according to any one of items 1-7, wherein (T)EW-7197 or a derivative thereof or a pharmaceutically acceptable salt or solvate thereof is present in the composition at a concentration of from about 0.001 mM to about 10 mM.

(Item 9)

A composition according to any one of items 1-8, wherein (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, is present in the composition at a concentration of from about 0.01 mM to about 5 mM.

(Item 10)

The composition according to any one of paragraphs 1-9, which is an eye drop.

(Item A1)

A method of treating or preventing a condition, disorder, or disease of the corneal endothelium in a subject, comprising administering to the subject an effective amount of (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridine- 6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof.

(Item A2)

The method of claim A1, wherein the method suppresses a decrease in the density of corneal endothelial cells.

(Item A3)

The method of A1 or A2, wherein the condition, disorder, or disease of the corneal endothelium is Fuchs' endothelial corneal dystrophy or endothelial corneal droplets.

(Item A4)

The method of A1, wherein the condition, disorder, or disease of the corneal endothelium results from overexpression of the extracellular matrix (ECM).

(Item A5)

The method of A4, wherein the extracellular matrix (ECM) is selected from the group consisting of type I collagen, type IV collagen, type V collagen, and fibronectin.

(Item A6)

The method of A4 or A5, wherein the condition, disorder, or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, droplet formation, Descemet's membrane thickening, corneal thickening, corneal opacity, scar, corneal opacity, corneal macula, leukoma, glare, and defocused vision.

(Item A7)

The method of any one of A1-A6, wherein the condition, disorder, or disease of the corneal endothelium is Fuchs' corneal endothelial dystrophy.

(Item A8)

The method of any one of A1-A7, wherein (T)EW-7197, or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, is administered at a concentration of from about 0.001 mM to about 10 mM.

(Item A9)

The method of any one of A1-A8, wherein (T)EW-7197, or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, is administered at a concentration of from about 0.01 mM to about 5 mM.

(Item A10)

The method of any one of A1-A9, wherein (T)EW-7197, or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, is administered as eye drops.

(Item B1)

Application of (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H -imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament for the treatment or prevention of a condition, disorder or disease of the corneal endothelium.

(Item B2)

Use according to item B1, wherein the drug is intended to suppress the decrease in the density of corneal endothelial cells.

(Item B3)

The use according to B1 or B2, wherein the condition, disorder or disease of the corneal endothelium is Fuchs endothelial corneal dystrophy or droplet endothelial corneal changes.

(Item B4)

The use of B1 wherein the condition, disorder or disease of the corneal endothelium results from overexpression of the extracellular matrix (ECM).

(Item B5)

The use of B4, wherein the extracellular matrix (ECM) is selected from the group consisting of type I collagen, type IV collagen, type V collagen, and fibronectin.

(Item B6)

The use of B4 or B5 wherein the condition, disorder or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, droplet formation, Descemet's membrane thickening, corneal thickening, corneal opacity, scar, corneal opacity, corneal macula, leukoma, glare and blurred vision.

(Item B7)

The use of any one of B1-B6 wherein the condition, disorder or disease of the corneal endothelium is Fuchs' corneal endothelial dystrophy.

(Item B8)

The use of any one of B1-B7 wherein (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, is present in the composition at a concentration of from about 0.001 mM to about 10 mM.

(Item B9)

The use of any one of B1-B8, wherein (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, is present in the drug at a concentration of from about 0.01 mM to about 5 mM.

(Item B10)

Use according to any one of B1-B9, wherein the drug is an eye drop.

(Item C1)

A compound for use in the treatment or prevention of a condition, disorder, or disease of the corneal endothelium, wherein the compound is (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridine- 6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof.

(Item C2)

A compound according to C1, wherein the compound is for use in suppressing a decline in corneal endothelial cell density.

(Item C3)

The compound of C1 or C2, wherein the condition, disorder or disease of the corneal endothelium is Fuchs endothelial corneal dystrophy or endothelial corneal droplets.

(Item C4)

The compound of C1 wherein the condition, disorder or disease of the corneal endothelium results from overexpression of the extracellular matrix (ECM).

(Item C5)

The compound of C4, wherein the extracellular matrix (ECM) is selected from the group consisting of type I collagen, type IV collagen, type V collagen, and fibronectin.

(Item C6)

The compound of C4 or C5, wherein the condition, disorder, or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, drop formation, Descemet's membrane thickening, corneal thickening, corneal opacity, scar, corneal opacity, corneal macula, leukoma, glare, and defocused vision.

(Item C7)

The compound of any one of C1-C6 wherein the condition, disorder or disease of the corneal endothelium is Fuchs' corneal endothelial dystrophy.

(Item C8)

A compound according to any one of C1-C7, wherein the compound is administered at a concentration of from about 0.001 mM to about 10 mM.

(Item C9)

A compound according to any one of C1-C8, wherein the compound is administered at a concentration of from about 0.01 mM to about 5 mM.

(Item C10)

A compound according to any one of C1-C9, wherein the compound is administered as eye drops.

[0006]

The present invention is intended to be able to provide one or more of the above features in additional combinations in addition to the combinations expressly shown. Other embodiments and advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description, as appropriate.

[Benefits of the present invention]

[0007]

The present invention relates to a medicament that treats or prevents a condition, disorder or disease of the corneal endothelium, comprising (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridine -6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof. Further, the present invention relates to a corneal endothelial cell preservation composition or a corneal endothelial cell growth promoting composition comprising (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof.

[Brief Description of Drawings]

[0008]

Fig. 1 shows phase-contrast microscopic images of immortalized corneal endothelial cells obtained from a patient with Fuchs' endothelial corneal dystrophy (iFECD), which is a model of corneal endothelial disease (left: without TGF-β supplementation, right: with TGF-β supplementation).

Fig. 2 shows phase contrast microscopic images of immortalized corneal endothelial cells from a patient with Fuchs' endothelial corneal dystrophy (iFECD) treated with EW-7197 and SB431542.

Fig. 3 shows a table summarizing the evaluation of the effect of suppression of cellular disorders in iFECD observed in figure 2 .

Fig. 4 shows phase contrast microscopic images of immortalized corneal endothelial cells obtained from a patient with Fuchs' endothelial corneal dystrophy (iFECD) treated with EW-7197 in a concentration dependent manner.

Fig. 5 shows graphs of cell survival and caspase 3/7 activity in iFECD in the presence of SB431542.

Fig. 6 shows graphs for percent cell survival and caspase 3/7 activity in iFECD in the presence of EW-7197.

Fig. 7 shows Western blot results for fibronectin, cleaved caspase 3 and PARP in iFECD.

Fig. 8 shows the results of type I collagen and fibronectin expression area analysis in the 0.02% EW-7197 eye drop group and vehicle eye drop group. The vertical axis indicates the expression area (in pixels).

Fig. 9 shows the results of the fibronectin expression area analysis in the 0.02% and 0.1% EW-7197 eye drop groups and vehicle eye drop group. The vertical axis indicates the expression area (in pixels). The left graph shows the results of the analysis after excluding 30,000 or less luminescence in the field of view at 40 times magnification (center, 6 o'clock and 9 o'clock (ear side)). The right graph shows the analysis results in the field of view at 100x magnification (3 o'clock direction (nose side)).

[Description of Embodiments]

[0009]

The present invention is described below. Throughout the specification, a singular expression is to be understood as encompassing its plural concept, unless specifically stated otherwise. Thus, singular articles (e.g. "a", "an", "the" and the like in the case of English) should also be understood to cover their plural concept unless specifically stated otherwise. In addition, the terms used in the present invention should be understood as used in the meaning that is usually used in the art, unless specifically indicated otherwise. Therefore, unless otherwise specified, all terms and scientific technical terms that are used in the present invention have the same meaning as commonly known to experts in the art to which the present invention pertains. In case of conflict, the present invention (including definitions) takes precedence.

[0010]

(Definitions)

As used in the present invention, "about" refers to a range of ±10% of the subsequent numerical value, unless specifically stated otherwise.

[0011]

As used in the present invention, "subject" refers to the target of administration (graft) of a therapeutic or preventive drug or method of the present invention. Examples of subjects include mammals (eg, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, and the like), but primates are preferred, and humans are particularly preferred.

[0012]

As used in the present invention, "EW-7197" and "TEW-7197" are used interchangeably, referring to N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl) -5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline. "EW-7197" and "TEW-7197" are also collectively referred to in the present invention as "(T)EW-7197".

[0013]

As used in the present invention, the condition, disorder or disease of the corneal endothelium "as a result of overexpression of extracellular matrix (ECM)" is mainly a condition, disorder or disease of the corneal endothelium associated with opacification, deposition, hypertrophy or the like of extracellular matrix, or a condition, which causes decreased vision due to drops on the surface of the corneal endothelium, thickening of the Descemet's membrane, such as cloudy drops of the Descemet's membrane, or the like. In corneal endothelial diseases such as Fuchs corneal dystrophy, overproduction of extracellular matrix impairs vision or visual recognition even without a decrease in cell numbers, in contrast to the exacerbation of a condition caused by death (especially apoptosis) of corneal endothelial cells. Thus, even if cell death can be suppressed, this needs to be addressed.

[0014]

As used herein, "derivative" refers to a compound with a chemical or physical modification, such as a functional group, that has the same or similar basic structure as the basic structure of the parent compound, but is different, or an additional functional group. The derivative has the same or similar biological activity as the parent compound.

[0015]

As used herein, a "pharmaceutically acceptable salt" refers to an inorganic or organic acid addition salt of a compound of the present invention that is relatively non-toxic. These salts can be prepared by reacting a temporarily purified compound between final isolation and purification of the compound or free base form alone with a suitable organic or inorganic salt and isolating the salt thus formed.

[0016]

Examples of pharmaceutically acceptable base salts of the compound of the present invention include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; aliphatic amine salts such as trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, procaine salts, meglumine salts, diethanolamine salts and ethylenediamine salts; aralkylamine salts such as N,N-dibenzylethylenediamine and benetamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts and isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salt, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts, tetrabutylammonium salts; basic amino acid salts such as arginine salts and lysine salts; and the like.

[0017]

Examples of pharmaceutically acceptable acid salts of the compound of the present invention include inorganic acid salts such as hydrochlorides, sulfates, nitrates, phosphates, carbonates, hydrogen carbonates and perchlorates; organic acid salts such as acetates, propionates, lactates, maleates, fumarates, tartrates, malates, citrates and ascorbate; sulfonates such as methanesulfonates, isethionates, benzenesulfonates and p-toluenesulfonates; acidic amino acids such as aspartates and glutamates; and the like.

[0018]

As used in the present invention, "solvate" refers to a solvate of a compound of the present invention or a pharmaceutically acceptable salt thereof, including, for example, a solvate of an organic solvent (e.g., an alcohol (ethanol or the like), a hydrate, etc. When a hydrate is formed, the compound may be coordinated with any the number of water molecules Examples of hydrates include monohydrates, dihydrates, and the like.

[0019]

As used in the present invention, "iFECD" (Fuchs' immobilized corneal endothelial dystrophy) is an abbreviation for immortalized Fuchs' corneal endothelial dystrophy cells.

[0020]

As used in the present invention, "HCEC" is an abbreviation for human corneal endothelial cells. In addition, "iHCEC" is an abbreviation for immortalized human corneal endothelial cells.

[0021]

As used in the present invention, "subject" refers to the target of administration (graft) of a therapeutic or preventive drug or method of the present invention. Examples of subjects include mammals (eg, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, and the like), but primates are preferred, and humans are particularly preferred.

[0022]

As used herein, "condition, disorder or disease of the corneal endothelium" refers to any condition, disorder or disease associated with the corneal endothelium. Representative examples thereof include, but are not limited to, Fuchs endothelial corneal dystrophy, droplet endothelial corneal changes, post-corneal transplant disorder, corneal endothelitis, trauma, post-ocular surgery disorder, post-laser eye surgery disorder, aging, posterior polymorphic dystrophy (PPD) , congenital hereditary endothelial dystrophy (CHED), idiopathic corneal endothelial disorder, cytomegalovirus corneal endothelitis, and the like. In a preferred embodiment, the condition, disorder, or disease of the corneal endothelium comprises Fuchs' corneal endothelial dystrophy. In another embodiment, the condition, disorder, or disease of the corneal endothelium results from overexpression of the extracellular matrix (ECM), for example, from overexpression of type I collagen, type IV collagen, type V collagen, and fibronectin.

[0023]

A condition, disorder or disease of the corneal endothelium resulting from overexpression of extracellular matrix (ECM) includes any condition, disorder or disease with overexpression of ECM seen in the corneal endothelium. Examples thereof include, but are not limited to, Fuchs endothelial corneal dystrophy, drop formation, Descemet's membrane thickening, corneal thickening, corneal opacity, scarring, corneal opacity, corneal macula, leukoma, glare, blurred vision, and the like.

[0024]

(General technology)

Molecular biology methodology, biochemical methodology and microbiological methodology used in the present invention are well known and traditionally used in the art, which are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd edition (2001); Ausubel, F.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M.A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. associates; Ausubel, F.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and yearly supplements; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R.L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996). Bioconjugate Techniques, Academic Press, Bessatsu Jikken Igaku [Experimental Medicine, Supplemental Volume] , Idenshi Donyu & Hatsugen Kaiseki Jikken Ho [Experimental Methods for Transgenesis & Expression Analysis] , Yodosha, 1997, or similar. Messages from Nancy Joyce et al. {Joyce, 2004 #161} and {Joyce, 2003 #7} are well known for corneal endothelial cells. However, as described above, long-term culture or subculture results in a fibroblast-like transformation, and research is ongoing for an efficient culture method. Their respective parts (which may be the whole document) are incorporated into the present invention by reference.

[0025]

(Description of Preferred Embodiments)

Preferred embodiments are described in the present invention below. It is clear that the embodiments are an example of the present invention, so the scope of the present invention is not limited to these preferred embodiments. It is clear that the person skilled in the art can refer to the following preferred embodiments, easily making modifications or changes within the scope of the present invention. Any of these embodiments may be combined as appropriate by those skilled in the art.

[0026]

<Composition>

In one aspect, the present invention relates to a composition for treating or preventing a condition, disorder or disease of the corneal endothelium, comprising (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a ]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof. In some embodiments, the composition of the present invention is designed to suppress a decrease in the density of corneal endothelial cells. (T)EW-7197 used in the composition of the present invention can suppress corneal endothelial disorders in the same manner as SB431542, which is known to suppress corneal endothelial disorders. Surprisingly, the effect of suppressing corneal endothelial disorders of (T)EW-7197 was also observed at very low concentrations. While the suppression effect of corneal endothelial disorders was not observed for SB431542 at sub-μM (1 μM to 0.1 μM), a very strong effect of suppression of corneal endothelial disorders was observed for (T)EW-7197 even at a low concentration of 0.03 µM. Thus, (T)EW-7197 used in the present invention can have a very high therapeutic effect on the corneal endothelium. In addition, cell toxicity was hardly detected in the cell survival test, so (T)EW-7197 is also excellent in terms of safety.

[0027]

In another aspect, the present invention relates to a composition for suppressing corneal endothelial cell density loss, comprising (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridine-6 -yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0028]

In another aspect, the present invention relates to (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridine -2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof (hereinafter referred to as a compound of the present invention or the like) for treating or preventing a condition, disorder or corneal endothelial disease. In some embodiments, the compound of the present invention, or the like, is intended to suppress a decline in corneal endothelial cell density. (T)EW-7197 in the compound of the present invention or the like can suppress corneal endothelial disorders in the same manner as SB431542, which is known to suppress corneal endothelial disorders. The effect of suppressing corneal endothelial disorders (T)EW-7197 was also observed at very low concentrations. While the suppression effect of corneal endothelial disorders was not observed for SB431542 at sub-μM (1 μM to 0.1 μM), a very strong effect of suppression of corneal endothelial disorders was observed for (T)EW-7197 even at a low concentration of 0.03 µM. Thus, (T)EW-7197 used in the present invention can have a very high therapeutic effect on the corneal endothelium. In addition, cell toxicity was hardly detected in the cell survival test, so (T)EW-7197 is also excellent in terms of safety.

[0029]

The composition of the present invention may be a pharmaceutical composition (eg eye drops, intracameral injection, intravitreal injection or subconjunctival injection). The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include, but are not limited to, any solvent, diluent, other liquid carriers, dispersion or suspending agents, surface activators, isotonizing agents, thickeners, emulsifiers, preservatives, solid binders, lubricating agents, and the like, as will be appropriate. for the specific dosage form required. Remington's Pharmaceutical Sciences, edited by Gennaro, Mack Publishing, Easton, PA, 1995, describes various carriers used in known techniques for preparing and preparing pharmaceutical compositions.

Some examples of materials that can function as a pharmaceutically acceptable carrier include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa powder and suppository wax; oil such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laureate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer solution. Other non-toxic and compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as color, release agent, coating agent, sweetener, flavor, flavor, preservative and antioxidant, may also be present in the composition according to the manufacturer's decision.

[0030]

In one embodiment, the composition of the present invention can treat or prevent a condition, disorder or disease of the corneal endothelium due to a decrease in corneal endothelial cell density. A condition, disorder or disease of the corneal endothelium due to a decrease in the density of corneal endothelial cells is selected from the group consisting of Fuchs corneal endothelial dystrophy, corneal drops, posterior polymorphic dystrophy, iridocorneal endothelial syndrome, congenital hereditary endothelial dystrophy, viral disease (cytomegalovirus corneal endothelitis or corneal endothelitis in herpes simplex virus), exfoliative syndrome, corneal transplant rejection, bullous keratopathy, corneal transplant disorder, corneal endotheliitis, trauma, eye surgery or laser eye surgery disorder, or aging.

[0031]

In one embodiment, the condition, disorder, or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, droplet endothelial corneal changes, corneal transplant disorder, corneal endothelitis, trauma, eye surgery disorder, laser eye surgery disorder , aging, posterior polymorphic dystrophy (PPD), congenital hereditary endothelial dystrophy (CHED), idiopathic corneal endothelial disorder, and cytomegalovirus corneal endothelitis. In another embodiment, the condition, disorder, or disease of the corneal endothelium is Fuchs' endothelial corneal dystrophy or droplet endothelial corneal changes.

[0032]

In another aspect, the present invention provides a composition for treating or preventing a corneal endothelial condition, disorder or disease resulting from extracellular matrix (ECM) overexpression, comprising (T)EW-7197 (N-((4-([1,2,4 ]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or its derivative, or pharmaceutically an acceptable salt, or solvate thereof. In another aspect, the present invention relates to (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridine -2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof (hereinafter referred to as a compound of the present invention or the like) for the treatment or prevention conditions, disorders or diseases of the corneal endothelium resulting from overexpression of the extracellular matrix (ECM). (T)EW-7197 can unexpectedly suppress the disruption (eg, overexpression) of an extracellular matrix (ECM), such as fibronectin, in corneal endothelial cells. For example, disruption (eg, overexpression) of an extracellular matrix (ECM) such as type I collagen, type IV collagen, or type V collagen is also confirmed in Fuchs endothelial corneal dystrophy. The composition of the present invention or the compound of the present invention or the like can also suppress the disturbance of these extracellular matrix (ECM) data. Examples of a corneal endothelial condition, disorder or disease resulting from extracellular matrix (ECM) overexpression include Fuchs endothelial corneal dystrophy, droplet formation, Descemet's membrane thickening, corneal thickening, corneal opacity, scar, corneal opacity, corneal macula, leukoma, glare, blurred vision and the like. Fuchs endothelial corneal dystrophy is a disease in which the cell density of the corneal endothelium is greatly reduced and extracellular matrix is deposited on the Descemet's membrane, resulting in corneal endothelial droplets and thickening of the Descemet's membrane. For this reason, the suppression of extracellular matrix overexpression implies that a significant improvement and, in some cases, a complete cure is possible in the treatment or prevention of Fuchs endothelial corneal dystrophy. It is also possible to improve, treat or prevent corneal endothelial droplet changes and thickening of the Descemet's membrane, as well as other conditions associated with opacification or deposition (irreversible opacification of the corneal stroma due to prolonged corneal edema or the like), which may occur due to overproduction of the extracellular matrix in violation of the corneal endothelium, such as Fuchs endothelial corneal dystrophy. In addition, overexpression of a proteoglycan such as agrin has also been confirmed in Fuchs endothelial corneal dystrophy. Overexpression of a proteoglycan such as agrin can lead to droplet endothelial changes in the cornea and thickening of the Descemet's membrane, as well as other conditions associated with opacification or deposition described above. The composition of the present invention or the compound of the present invention or the like can also suppress the overexpression of a proteoglycan such as agrin.

[0033]

In one embodiment, examples of the method of using the present invention include, but are not limited to, eye drops. Other examples thereof include dosing regimens (modes of administration and dosage forms) such as ophthalmic ointment, intracameral injection, impregnation into a sustained release agent, subconjunctival injection, systemic administration (oral administration, intravenous injection), and the like.

[0034]

The concentration of (T)EW-7197 used in the present invention is usually from 0.001 to about 100 µM (µmol/L), preferably from about 0.01 to about 30 µM, more preferably from about 0.03 to about 10 µM, and even more preferably from about 0.03 to about 1 μm. Examples of other concentration ranges include, but are not limited to, typically about 0.01 nM to about 100 µM, about 0.1 nM to about 100 µM, about 0.001 to about 100 µM, about 0.01 to about 75 µM , from about 0.05 to about 50 µM, from about 1 to about 10 µM, from about 0.01 to about 10 µM, from about 0.05 to about 10 µM, from about 0.075 to about 10 µM, from about 0 .1 to about 10 µM, about 0.5 to about 10 µM, about 0.75 to about 10 µM, about 1.0 to about 10 µM, about 1.25 to about 10 µM, about 1 .5 to about 10 µM, about 1.75 to about 10 µM, about 2.0 to about 10 µM, about 2.5 to about 10 µM, about 3.0 to about 10 µM, about 4 0 to about 10 µM, about 5.0 to about 10 µM, about 6.0 to about 10 µM, about 7.0 to about 10 µM, about 8.0 to about 10 µM, about 9 0 to about 10 µM, about 0.01 to about 50 µM, about 0.05 to about 5.0 µM, about 0.075 to about 5.0 µM, about 0.1 to about 5.0 µM , from about 0.5 to about 5.0 µM, from about 0.75 to about 5.0 µM, from about 1.0 to about 5.0 µM, from about 1.25 to about 5.0 µM, from about 1.5 to about 5.0 µM, about 1.75 to about 5.0 µM, about 2.0 to about 5.0 µM, about 2.5 to about 5.0 µM, about 3 0 to about 5.0 µM, about 4.0 to about 5.0 µM, about 0.01 to about 3.0 µM, about 0.05 to about 3.0 µM, about 0.075 to about 3.0 µM, about 0.1 to about 3.0 µM, about 0.5 to about 3.0 µM, about 0.75 to about 3.0 µM, about 1.0 to about 3, 0 µM, about 1.25 to about 3.0 µM, about 1.5 to about 3.0 µM, about 1.75 to about 3.0 µM, about 2.0 to about 3.0 µM , from about 0.01 to about 1.0 µM, from about 0.05 to about 1.0 µM, from about 0.075 to about 1.0 µM, from about 0.1 to about 1.0 µM, from about 0 .5 to about 1.0 µM, from about 0.75 to about 1.0 µM, from about 0.09 to about 35 µM, and from about 0.09 to about 3.2 µM, more preferably from about 0, 01 to about 10 µM, from about 0.1 to about 3 µM, and from about 0.1 to about 1.0 µM.

[0035]

When applied as eye drops, the concentration in the formulation can be determined to be about 1-10000 times, preferably about 100-10000 times, such as about 1000 times the effective concentration described above, as a reference value, taking into account the dilution of the lacrimal fluid and taking toxicity into account. The concentration can be set to a concentration greater than this concentration. Examples thereof include from about 0.01 µM (µmol/L) to about 1000 mM (mmol/L), from about 0.03 µM to about 1000 mM, from about 0.1 µM to about 100 mM, from about 0, 3 µM to about 100 mM, from about 1 µM to about 100 mM, from about 3 µM to about 100 mM, from about 10 µM to about 100 mM, from about 30 µM to about 100 mM, from about 0.1 µM to approximately 30 mM, approximately 0.3 μM to approximately 30 mM, approximately 1 μM to approximately 30 mM, approximately 3 μM to approximately 30 mM, approximately 1 μM to approximately 10 mM, approximately 3 μM to approximately 10 mM , from about 10 µM to about 1 mM, from about 30 µM to about 1 mM, from about 10 µM to about 10 mM, from about 30 µM to about 10 mM, from about 100 µM to about 10 mM, from about 300 µM up to about 10 mM, from about 10 µM to about 100 mM, from about 30 µM to about 100 mM, from about 100 µM to about 100 mM, from about 300 µM to about 100 mM, from about 1 mM to about 10 mM, from about 1 mM to about 50 mM; and from about 1 mM to about 100 mM. These upper limits and lower limits can be combined and defined as appropriate.

[0036]

In one embodiment, (T)EW-7197, or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, may be present in the composition at a concentration of from about 0.03 µM to about 10 µM, and preferably from about 0.03 µM to about 1 μM.

[0037]

In still another embodiment, the composition of the present invention is provided as eye drops, wherein (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof may be present at about 0.03 mM to about 100 mM, preferably from about 0.001 mM to about 10 mM, more preferably from about 0.05 mM to about 10 mM, even more preferably from about 0.01 mM to about 5 mM, and most preferably from about 0.1 mM to about 5 mM. In a particular embodiment, a composition of the present invention provided as eye drops may contain about 0.5 mM (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0038]

The effective dose of a drug of the present invention that is effective for the treatment of a particular disease, disorder or condition may vary depending on the nature of the disorder or condition, but may be determined by a person skilled in the art using a standard clinical method based on the description provided in the present invention. . In addition, an in vitro assay may be used to assist in determining an optimal dosage range, if necessary. Since the exact dose to be used in the combination agent may also vary depending on the route of administration and the severity of the disease or disorder, the dose should be determined according to the judgment of the physician or the condition of each patient. However, the dose, although not specifically limited, may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight per dosage, or a value in the range of any two of these values. The dosing interval is not particularly limited, but may be, for example, 1 or 2 administrations over 1, 7, 14, 21, or 28 days, or 1 or 2 administrations per day in the range of any two of them. The dosage, the number of doses, the dosing interval, the dosing period, and the dosing method can be appropriately selected depending on the patient's age or body weight, condition, dosing regimen, target organ, and the like. For example, the present invention may be administered as eye drops. In addition, the drug of the present invention can be administered into the anterior chamber. In addition, the therapeutic drug contains the active ingredient in a therapeutically effective amount or in an amount effective to produce the desired effect. When the therapeutic marker is significantly reduced after administration, it can be determined that a therapeutic effect has been exerted. An effective dose can be estimated from a dose-response curve obtained from an in vitro testing system or an animal model.

[0039]

<Therapeutic or preventive method>

In one aspect, the present invention is a method of treating or preventing a corneal endothelial condition, disorder, or disease in a subject, comprising administering to the subject an effective amount of (T)EW-7197 (N-((4-([1,2,4]triazolo[ 1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or its solvate. One or more of the embodiments described above in the <Composition> paragraph of the present invention, or the like, may be suitably used in the method of the present invention.

[0040]

In yet another aspect, the present invention is a method of treating or preventing a corneal endothelial condition, disorder or disease resulting from extracellular matrix (ECM) overexpression, comprising administering to a subject an effective amount of (T)EW-7197 (N-((4-([1 ,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or its derivative , or a pharmaceutically acceptable salt thereof, or a solvate thereof. One or more of the embodiments described above in the <Composition> paragraph of the present invention, or the like, may be suitably used in the method of the present invention.

[0041]

<Application>

In one aspect, the present invention relates to the use of (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6- methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof in the preparation of a medicament for the treatment or prevention of a condition, disorder or disease of the corneal endothelium . One or more of the embodiments described above in the <Composition> paragraph of the present invention, or the like, may be suitably used in the practice of the present invention.

In yet another aspect, the present invention relates to the use of (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6 -methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament for the treatment or prevention of an endothelial condition, disorder or disease cornea as a result of extracellular matrix (ECM) overexpression. One or more of the embodiments described above in the <Composition> paragraph of the present invention, or the like, may be suitably used in the practice of the present invention.

[0042]

<Preservation Composition and Preservation Method>

In another aspect, the present invention relates to a corneal endothelial cell preservation composition comprising (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof. In a preferred embodiment, the preservation is cryopreservation. It is clear that (T)EW-7197 used in the present invention can be used in any form described in the present invention, such as a form that is suitable as a preservation composition among the embodiments described as a medicine. As used herein, a "preservation composition" is a composition for preserving a corneal fragment obtained from a donor for a period of time before the fragment is transplanted into a recipient, or for preserving a corneal endothelial cell prior to growth or cultivation of a corneal endothelial cell.

[0043]

In one aspect, the present invention relates to the use of (T)EW-7197, or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a corneal endothelial cell preservation composition. In another aspect, the present invention provides (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, for use in corneal endothelial cell preservation. In yet another aspect, the present invention relates to a method for preserving a corneal endothelial cell, comprising preserving using a composition for preserving a corneal endothelial cell comprising (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0044]

In one embodiment, a preservative composition of the present invention can be prepared by adding (T)EW-7197 of the present invention to a commonly used preservative agent or preservative solution. Examples of this corneal preservation solution include preservation solutions that are commonly used for corneal graft (Sclera-Cornea Fibrous Eye Fragment Preservation Solution (Optisol GS®) or Corneal Graft Eyeball Preservation Solution (EPII®) ), saline, phosphate buffered saline (PBS), and the like.

[0045]

The preservation composition of the present invention is used for preservation of the cornea, which is used in organ transplantation or the like. The preservation composition of the present invention is also used as a preservation solution for cryopreservation of a corneal endothelial cell or as a component thereof.

[0046]

In another embodiment of a preservation composition of the present invention used for cryopreservation, an existing cryopreservation solution may be used by adding a preservation composition comprising (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof of the present invention. Examples of the cryopreservation solution include, but are not limited to, the CELLBANKER® series provided by Takara Bio (CELL BANKER PLUS (Part No: CB021), CELL BANKER 2 (Part No: CB031), STEM-CELLBANKER (Part No: CB043) and the like) , KM BANKER (Kohjin Bio, catalog number: KOJ-16092005), and Freezing Medium, Animal Component Free, CRYO Defined (also listed as Cnt-CRYO) (CELLNTEC, catalog number: CnT-CRYO-50). In yet another embodiment, the cryopreservation solution used is KM BANKER. It is clear that a person skilled in the art can apply a suitably modified cryopreservation solution by suitably changing the constituent of the cryopreservation solution described above, or by adding an additional constituent. Glycerin, dimethyl sulfoxide, polyethylene glycol, acetamide or the like can be further added to the preservation solution of the present invention for cryopreservation.

[0047]

Reference literature, such as scientific literature, patents and patent applications cited in the present invention, are incorporated into the present invention by reference to the same extent that the full text of each document is specifically described.

[0048]

As described above, the present invention has been described, showing the preferred embodiments for ease of understanding. The present invention is described below based on examples. The above description and the following examples are not intended to limit the present invention, but are intended to be exemplary only. Thus, the scope of the present invention is not limited to the embodiments shown in the examples specifically described in the present invention, and is limited only by the scope of the claims.

[Examples]

[0049]

The following describes examples of the present invention. Biological samples or the like, where applicable, have been handled in accordance with the standards adopted by the Ministry of Health, Labor and Welfare, the Ministry of Education, Culture, Sports, Science and Technology and the like and, where applicable, on the basis of the Declaration of Helsinki or codes of ethics prepared on their basis. Regarding the donation of eyes used for the study, consent was obtained from the next of kin of all deceased donors. This study was approved by the Ethics Committee of the University of Erlangen-Nuremberg (Germany) and the SightLife™ Eye Bank (Seattle, Washington) or its respective body.

[0050]

(Production Example: Obtaining an Immortalized Line Model (iFECD) of Corneal Endothelial Cells Obtained from a Patient with Fuchs Endothelial Corneal Dystrophy)

In this example, an immortalized corneal endothelial cell line (iFECD) was obtained from corneal endothelial cells from patients with corneal endothelial dystrophy.

[0051]

(Cultivation method)

Corneal endothelial cells were mechanically separated with a basement membrane from the cornea for examination, purchased from the Seattle Eye Bank. After collagenase was used to separate and harvest corneal endothelial cells from the basement membrane, the cells were subjected to primary culture. As for the medium, Opti-MEM I Reduced-Serum Medium, Liquid (INVITROGEN cat. no.: 31985-070) to which was added 8% FBS (BIOWEST, cat. no.: S1820-500), 200 mg/mL CaCl ₂ ·2H ₂ O (SIGMA catalog number: C7902-500G), 0.08% chondroitin sulfate (SIGMA catalog number: C9819-5G), 20 µg/ml ascorbic acid (SIGMA catalog number: A4544-25G), 50 µg/ml gentamicin (INVITROGEN catalog number: number: 15710-064) and 5 ng/ml EGF (INVITROGEN catalog number: PHG0311), and conditioned for 3T3 feeder cells, used as basal medium. In addition, cells were grown in basal medium supplemented with SB431542 (1 μmol/L) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5(4-pyridyl)imidazole-4-[4 -(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine) (1 µmol/l) (also referred to in the present invention as "SB203580+SB431542+3T3 conditioned medium").

[0052]

(collection method)

Corneal endothelial cells were obtained with the approval of the ethics committee and the written consent of 3 patients who suffered from bullous keratopathy in accordance with the clinical diagnosis of Fuchs endothelial corneal dystrophy and underwent corneal endothelial transplantation (Descemet's membrane endothelial keratoplasty=DMEK). For DMEK, pathological corneal endothelial cells were mechanically peeled off with basement membrane, ie, Descemet's membrane, and immersed in Optisol-GS corneal preservation solution (Bausch & Lomb). Then, collagenase treatment was applied to enzymatically harvest corneal endothelial cells, and the cells were cultured in conditioned medium SB203580+SB431542+3T3. For cultured corneal endothelial cells from a patient with Fuchs endothelial corneal dystrophy, the SV40 large T antigen and the hTERT gene were amplified by PCR and introduced into a lentiviral vector (pLenti6,3_V5-TOPO; Life Technologies Inc.). The lentiviral vector was then used to infect 293T cells (RCB2202; Riken Bioresource Center, Ibaraki, Japan) with a transfection reagent (Fugene HD; Promega Corp., Madison, WI) and three types of helper plasmids (pLP1, pLP2, pLP/VSVG; Life Technologies Inc.). The culture supernatant containing the viruses was collected 48 hours after infection. 5 μg/ml polybrene was used and added to the culture solution of cultured corneal endothelial cells of a patient with Fuchs endothelial corneal dystrophy, and the SV40 large T antigen and the hTERT gene were introduced. Images of the immortalized corneal endothelial cell line (iFECD) of patients with Fuchs endothelial corneal dystrophy were studied under a phase-contrast microscope. Cultured corneal endothelial cells from the test cornea, imported from the Seattle Eye Bank, were immortalized in the same manner to obtain an immortalized normal corneal endothelial cell line (iHCEC) as a control. When examining images of an immortalized endothelial cell line (iFECD) and an immortalized endothelial cell line from a healthy donor (iHCEC) under a phase-contrast microscope, both iHCEC and iFECD had a polygonal-shaped layer, as in normal corneal endothelial cells. iHCEC and iFECD were maintained and cultured in Dulbecco's Modified Eagle's Medium (DMEM)+10% fetal bovine serum (FBS).

[0053]

(Example of observation with a phase contrast microscope)

The medium was removed from the culture dish in which the immortalized human corneal endothelial cells of a patient with Fuchs endothelial corneal dystrophy (iFECD) were cultured and added and washed with 1×PBS (-) preheated to 37°C. This was repeated twice. 1×PBS (-) was added again and the medium was incubated for 5 minutes at 37°C (5% CO ₂ ). After removal of PBS (-), 0.05% trypsin-EDTA (Nacalai Tesque, 32778-34) was added and incubated for 5 minutes at 37°C (5% CO ₂ ). The medium was then suspended and centrifuged for 3 minutes at 1500 rpm to recover the cells. DMEM (Nacalai Tesque, 08456-36) + 10% FBS (Biowest, S1820-500) + 1% P/S (Nacalai Tesque, 26252-94) was used as the medium.

[0054]

Immortalized human corneal endothelial cells from a patient with Fuchs Endothelial Corneal Dystrophy (iFECD) were seeded in a 12-well plate at a ratio of 8.0×10 ⁴ cells per well and cultured for 24 hours at 37°C (5% CO ₂ ). DMEM+10% FBS+1% P/S was used as the medium.

[0055]

After 24 hours, the medium was changed and cultured again for 24 hours. DMEM+2% FBS+1% P/S was used as the medium.

[0056]

After 24 hours, the medium was removed. 10 ng/ml human recombinant transforming growth factor-β2 (WAKO, 200-19911) was added and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, cell morphology and apoptosis were observed under a phase contrast microscope.

[0057]

Immortalized human corneal endothelial cells from a patient with Fuchs' endothelial corneal dystrophy (iFECD) were damaged when the cells were stimulated with TGF-β, as shown in Figure 1.

[0058]

Example 1 EW-7197 Corneal Endothelial Cell Disorder Suppressing Effect

In this example, the effect of suppressing corneal endothelial cell disorders EW-7197 was observed using immortalized human corneal endothelial cells from a patient with Fuchs' corneal endothelial dystrophy (iFECD). The cells were observed in a manner based on the observation example described above. The target was SB431542, which is known to inhibit corneal endothelial cell disorders (see International Publication No. WO 2015/064768).

[0059]

(Materials and methods)

After the iFECD was plated on a 12-well plate and cultured for 24 hours, the medium was removed. SB431542 (WAKO, 192-16541) and EW-7197 (Selleck Chemicals, S7530) were added so that the final concentration of each was 0.1, 0.3, 1, 3, or 10 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium.

[0060]

After 24 hours, the medium was removed. 10 ng/ml of human recombinant transforming growth factor-β2 (WAKO, 200-19911), as well as SB431542 and EW-7197, was added so that the final concentration of each was 0.1, 0.3, 1, 3, or 10 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium.

[0061]

After 24 hours, cell morphology and apoptosis were observed on a phase contrast microscope.

(Results)

The results are shown in Figures 2 and 3. It was observed that with pre-treatment with SB431542, the disorder of corneal endothelial cells was suppressed at 3 μM and 10 μM, especially at 10 μM, and the disorder of corneal endothelial cells was effectively suppressed, especially at 10 μM. Meanwhile, it was observed that by pre-treatment with EW-7197, the disorder of corneal endothelial cells was effectively suppressed, especially at 0.1, 0.3 and 1 μM. The effect of suppressing the disorder of corneal endothelial cells was also confirmed at 3 μM and 10 μM. It was unexpectedly found that EW-7197 can suppress corneal endothelial cell disorders even at very low concentrations compared to SB431542, which is known to be able to suppress corneal endothelial cell disorders.

[0062]

Example 2: EW-7197 Concentration Study

In this example, the concentration of EW-7197 was studied at which the effect of suppressing corneal endothelial cell disorders was effectively observed. The suppression effect of corneal endothelial cell disorders was observed in the same manner as in Example 1.

[0063]

(Materials and methods)

After iFECD was seeded on a 12-well plate at a ratio of 7.0×10 ⁴ cells and cultured for 24 hours, the medium was removed. EW-7197 (Selleck Chemicals, S7530) was added so that the final concentration was 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium.

[0064]

After 24 hours, the medium was removed. 10 ng/ml human recombinant transforming growth factor-β2 (WAKO, 200-19911) and EW-7197 were added so that the final concentration was 0.001, 0.003, 0.01, 0.03, 0.1, 0, 3, 1 , 3, 10, or 30 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium.

[0065]

After 24 hours, cell morphology and apoptosis were observed on a phase contrast microscope.

[0066]

(Results)

The results are shown in figure 4 . Significant cell damage was observed when immortalized human corneal endothelial cells from a patient with Fuchs' endothelial corneal dystrophy (iFECD) stimulated TGF-β. In the EW-7197 addition group, a particularly excellent suppression effect on corneal endothelial cell disorders was observed, especially at 0.03, 0.1, 0.3 and 1 μM. It was also confirmed that corneal endothelial cell disorders were effectively suppressed at 3 μM and 10 μM. Thus, it was found that EW-7197 effectively suppresses corneal endothelial cell disorders in a wide range of concentrations.

[0067]

Example 3 Cell Survival Rate and Caspase 3/7 Activity

In this example, cell viability and caspase 3/7 activity are analyzed in the presence of EW-7197. SB431542 was used as a control for comparison.

[0068]

(Materials and methods)

(cell viability analysis)

iFECD were seeded on a 96-well plate at a ratio of 3×10 ³ cells per well and cultured to achieve confluence at 37°C (5% CO ₂ ). DMEM+10% FBS+1% P/S was used as the medium.

[0069]

After 24 hours, the medium was removed. SB431542 (WAKO, 192-16541) and EW-7197 (Selleck Chemicals, S7530) were added so that the final concentration of each was 0.01, 0.1, 1, or 10 μM, and the medium was cultured for 48 hours. DMEM+2% FBS+1% P/S was used as the medium.

[0070]

After 24 hours, cell morphology was observed under a phase contrast microscope.

[0071]

After observation, cell viability was analyzed using Cell Titer-Glo fluorescent cell viability assay using the following methods. The medium was removed so that the amount was 50 μl per well, and Cell Titer-Glo Fluorescent Cell Viability Assay Solution (Promega, G7572) was added at 50 μl/well so that the ratio to medium was 1:1. Subsequently, the method was carried out without exposure to light. The mixture was thoroughly mixed using a shaker for 2 minutes at approximately 120 minutes ^-1 and incubated for 10 minutes. After incubation, 80 μl was transferred to an assay plate (Corning, 3912, 96 well assay plate, white polystyrene) and absorbance was measured using a GloMax-Multi detection system (Promega, E7051).

[0072]

(Caspase 3/7 analysis)

iFECD were seeded on a 96-well plate at a ratio of 3×10 ³ cells per well and cultured to achieve confluence at 37°C (5% CO ₂ ). DMEM+10% FBS+1% P/S was used as the medium.

[0073]

After 24 hours, the medium was removed. SB431542 (WAKO, 192-16541) and EW-7197 (Selleck Chemicals, S7530) were added so that the final concentration of each was 0.01, 0.1, 1, or 10 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium.

[0074]

[0075]

After 24 hours, cell morphology was observed under a phase contrast microscope.

[0076]:

After observation, caspase 3/7 activity was measured using the Caspase-Glo 3/7 assay using the following methods. The medium was removed so that the amount was 50 μl per well, and the Caspase Glo 3/7 assay reagent solution (mixture of Caspase-Glo 3/7 assay buffer and Caspase-Glo 3/7 assay substrate) (Promega, G8091) was added at 50 µl/well so that its ratio to the medium is 1:1. Then, the method was carried out without exposure to light. The mixture was thoroughly mixed using a shaker for 2 minutes at approximately 120 minutes ^-1 and incubated for 40 minutes at room temperature. After incubation, 80 µl was transferred to an assay plate (Corning, 3912, 96 well assay plate, white polystyrene) and absorbance was measured using the GloMax-Multi detection system (Promega, E7051)

[0077]

A caspase inhibitor, ie, 10 μM MZ-VD-FMK (WAKO, 262-0206I) was used as a positive control.

[0078]

(Results)

(cell viability analysis)

The results are shown in figures 5 and 6 . As a result of measuring the cell survival rate using the Cell Titer-Glo luminescent cell viability assay, no significant difference was found in the cell survival rate when SB431542 was added compared to the control group at all concentrations of 0.01, 0.1, 1 and 10 μM. In view of the above, the addition of SB431542 did not disrupt cells at concentrations of 0.01, 0.1, 1, and 10 μM. Similarly, no significant difference was found in cell survival when EW-7197 was added compared to the control group at all concentrations of 0.01, 0.1, 1 and 10 μM. In view of the above, the addition of EW-7197 does not disrupt cells at concentrations of 0.01, 0.1, 1, and 10 μM.

[0079]

(caspase 3/7 assay)

The results are shown in figures 5 and 6 . The Caspase-Glo 3/7 assay can measure the activity of caspase 3/7 associated with the induction of apoptosis. Specifically, higher caspase 3/7 activity indicates that a cellular disorder is being induced. From figure 5 it was found that when stimulated with TGF-β caspase 3/7 is significantly activated compared with no stimulation. Meanwhile, when SB431542 was added, caspase 3/7 activity decreased in the range of 0.01, 0.1, 1, and 10 µM. In particular, at 10 μM the value was similar to that in the control group without TGF-β stimulation. Accordingly, SB431542 inhibits caspase 3/7 activity in a concentration dependent manner. Its effect is most effective at 10 µM. When EW-7197 was added, caspase 3/7 was significantly inhibited at 0.01 μM. The value was similar to the control group without sham with TGF-β, especially at 0.1, 1 and 10 μM. It was found that EW-7197 can also inhibit caspase 3/7 in the same way as SB431542. In particular, a higher inhibitory effect was observed for EW-7197 compared to SB431542 even at 0.1 μM.

[0080]

Example 4: Western blot

In this example, expression of fibronectin (about 240 kDa), activated cleaved caspase 3 (about 17 kDa) and PARP (about 89 kDa) was tested in iFECD when EW-7197 was added by Western blot.

[0081]

(Materials and methods)

iFECD were seeded on a 12-well plate at a ratio of 8×10 ⁴ cells per well and cultured at 37°C (5% CO ₂ ) for 24 hours. DMEM+10% FBS+1% P/S was used as the medium.

[0082]

After 24 hours, the medium was removed. EW-7197 (Selleck Chemicals, S7530) was added so that the final concentration was 0.1, 1, or 10 μM, and SB431542 (WAKO, 192-16541) was added so that the final concentration was 10 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium.

[0083]

After 24 hours, the medium was removed and cultured for 24 hours so that the final concentration of 10 ng/ml human recombinant transforming growth factor-β2 (WAKO, 200-19911) and EW-7917 was 0.1, 1, and 10 μM, and the final concentration of SB431542 was 10 μM. DMEM+2% FBS+1% P/S was used as the medium.

[0084]

After 24 hours, cell morphology and apoptosis were observed on a phase contrast microscope.

[0085]

After observation, a Western blot was performed on the proteins using the following methods.

[0086]

1) protein extraction

To recover floating and dead cells, the medium was taken out on ice, the cells were washed twice with 1×PBS (-) and their solution was also taken out. The solution was centrifuged for 5 min at 4° C. and 800 g and the supernatant was discarded to give a pellet.

[0087]

Protein extraction buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) was added to washed cells for ice for protein extraction.

[0088]

Then, the above-described sediment of floating and dead cells after centrifugation was also suspended and taken out together. The recovered solution was crushed three times for 30 seconds in chilled water using an ultrasonicator (BIORUPTOR, TOSHO DENKI), and then centrifuged for 10 minutes at 4° C. and 15,000 rpm. The protein supernatant was recovered.

[0089]

2) Western blotting

The extracted protein was separated by SDS-PAGE and transcribed onto a nitrocellulose membrane. The amount of protein was 4 µg for fibronectin, PARP and GAPDH, and 10 µg for caspase 3. Mouse anti-fibronectin antibody (BD Biosciences, 610077), rabbit anti-caspase 3 antibody (Cell Signaling, 9662), rabbit anti-PARP antibody ( Cell signaling, 9542) and mouse anti-GAPDH antibody (MBL, M171-3) were used as the primary antibody.

[0090]

As a secondary antibody, a peroxidase-labeled anti-rabbit antibody and an anti-mouse antibody (GE Healthcare Biosciences, NA931V, NA934V) were used. For the primary antibody, mouse anti-fibronectin antibody was diluted 20,000-fold, rabbit anti-PARP antibody was diluted 1,000-fold, rabbit anti-caspase 3 antibody was diluted 1,000-fold, and mouse anti-GAPDH antibody was diluted 3,000-fold. All secondary antibodies were diluted 5000-fold. Chemi Lumi ONE Ultra (Nacalai Tesque, 11644-40) was used for detection. The detected band intensities were analyzed with a Lumino Image analyzer LAS-4000 mini (Fuji Film) and ImageQuantTM software (GE Healthcare).

[0091]

(Results)

The results are shown in figure 7 . When iFECD was stimulated with TGF-β, expression of fibronectin (about 240 kDa), activated form of cleaved caspase 3 (about 17 kDa) and PARP (about 89 kDa) was observed. Meanwhile, fibronectin expression was suppressed to the same extent as in the control group without TGF-β stimulation or 10 μM SB431542 at any concentration in the presence of EW-7197. The activated form of cleaved caspase 3 and PARP activity were also suppressed to the same extent as in the control group without stimulation with TGF-β or 10 μM SB431542. Therefore, Western blot analysis showed that EW-7197 represses fibronectin expression in the same way as SB431542.

[0092]

Example 5 Evaluation of EW-7197 Eye Drop Drug Efficacy in Fuchs Endothelial Corneal Dystrophy Using Col8a2 Knockin Mice

In this example, the efficacy of EW-7197 eye drops in Fuchs endothelial corneal dystrophy was evaluated using an animal model of Fuchs endothelial corneal dystrophy mice with Col8a2 knockin. Col8a2 knockine mice are established animal models of Fuchs endothelial corneal dystrophy (ref: Jun et al., Hum Mol Genet. 2012 Jan 15; 21(2): 384-93).

[0093]

5 μl of 0.02% (approximately 0.5 mM) EW-7197 eye drops or base agent (PBS containing 7% DMSO) were administered as eye drops to both eyes of 4-month-old mice, knockin at the alpha 2 gene of collagen 8 (Col8a2), 4 times a day for 8 weeks. Corneal endothelial cell density was measured using a KS3M mirror microscope (Konan Medical, Inc.) before the eye drops and 2, 4, 6 and 8 weeks after the eye drops. Mice were sacrificed by intraperitoneal administration of an excessive amount (200 mg/kg body weight or more) of an injection of the anesthetic somnopentyl (Kyoritsu Seiyaku Corporation) 8 weeks after the injection. After cardiac arrest was confirmed, both eyeballs were harvested. The collected eyeballs were incised to obtain a fragment of the cornea. Immunostaining of type I collagen and fibronectin was performed as follows. Image-Pro (Nippon Roper K.K.) was used to analyze the area of expression of type I collagen and fibronectin.

[0094]

(method for immunostaining type I collagen and fibronectin)

After immobilizing the mouse cornea fragment for 10 minutes at room temperature in 4% paraformaldehyde, 0.5% TritonX-100 was added and allowed to infiltrate for 5 minutes. Added 1% bovine serum albumin (BSA), and the fragment was incubated for 1 hour at room temperature. The corneal fragment was then immersed in the primary antibody solution and reacted overnight at 4°C. As the primary antibody, a 1:200 dilution of type I collagen polyclonal antibody (Rockland Immunochemicals) and a 1:250 dilution of fibronectin polyclonal antibody (abcam) were used. Then, the cornea fragment was immersed in a mixture of secondary antibody solution and DAPI solution (Dojindo Laboratories), and they reacted for 1 hour at room temperature away from light. A 1:2000 dilution of Alexa Fluor® 488-labeled goat anti-rabbit IgG (Life Technologies) was used as a secondary antibody. A diluted 1:1000 DAPI solution was used. The corneal fragment was stretched and placed on a glass slide and covered with a coverslip. The prepared sample was observed using an Olympus FV1000 FLUOVIEW confocal laser scanning microscope (Olympus Corporation).

[0095]

(Results)

Table 1 shows the magnitude of change in corneal endothelial cell density starting with eye drops up to 4 weeks and 8 weeks after eye drops in Col8a2 knockin mice (ΔECD (cells/mm ² )). A decrease in corneal endothelial cell density of 51.5 cells/mm ² was observed after 4 weeks, and a decrease in corneal endothelial cell density of 123.6 cells/mm ² was observed after 8 weeks in normal mice without the introduction of the Col8a2 gene. It is clear that the slight decrease in corneal endothelial cell density in normal mice is a consequence of the decline in corneal endothelial cells due to aging.

[Table 1]

Table 1. Magnitude of change in corneal endothelial cell density in Col8a2 knockin mice (ΔECD (cells/mm ² ))

a week 0 (before eye drops) 4 8 Foundation agent 0 -227.667 -401.889 0.02% T.E.W. 0 -121.4 -284.5

[0096]

As evident from Table 1, the reduction in corneal endothelial cell density in the 0.02% EW-7197 eye drop administration group was significantly suppressed compared to the base agent administration group. A significant increase in the expression of type I collagen and fibronectin was observed by immunostaining in Col8a2 knockin mice. Figure 8 shows the results of the analysis of the area of expression of type I collagen and fibronectin. Expression of type I collagen and fibronectin tended to decrease in the 0.02% EW-7197 eye drop group compared to the base agent group.

[0097]

Thus, it was found that EW-7197 migrated sufficiently to the corneal endothelium and effectively suppressed the decrease in the number of corneal endothelial cells (apoptosis of corneal endothelial cells and the like) and/or corneal endothelial disorders represented by overexpression of extracellular matrix (collagen type I, fibronectin and the like) with EW-7197 eye drops.

[0098]

Example 6 Evaluation of EW-7197 Eye Drops at Different Concentrations on Fuchs Endothelial Corneal Dystrophy Using Col8a2 Knockin Mice

In this example, the efficacy of EW-7197 eye drops at various concentrations on Fuchs endothelial corneal dystrophy was evaluated using Col8a2 knockin mice.

[0099]

(Way)

5 µl of 0.02% (approximately 0.5 mM) EW-7197 eye drops, 0.1% (approximately 2.5 mM) EW-7197 eye drops, or base agent (phosphate buffer containing DMSO) was administered as eye drops in both eyes of 4-month-old mice, with knockin of the alpha 2 collagen 8 gene (Col8a2), 4 times a day for 12 weeks. The surfactant was added to the base agent as needed. A person skilled in the art can use a suitable and suitable surfactant (Kenji Motose (1984). "Tenganzai" [ Eye drops ] Nanzando; International Pharmaceutical Excipients Council Japan (2016). "Iyakuhin Tenkabutsu Jiten" [ Pharmaceutical Excipient Encyclopedia ] Yakuji Nippo , Limited). Corneal endothelial cell density was measured with a KS3M mirror microscope (Konan Medical, Inc.) before the eye drops and 3, 6, 9 and 12 weeks after the eye drops. Mice were sacrificed by intraperitoneal administration of an excessive amount (200 mg/kg body weight or more) of somnpentyl anesthetic injection (Kyoritsu Seiyaku Corporation) 12 weeks after injection. After cardiac arrest was confirmed, both eyeballs were harvested. The collected eyeballs were cut to obtain a fragment of the cornea. Fibronectin immunostaining was performed. The method of immunostaining was as follows. Image-Pro (Nippon Roper KK) was used to analyze the region of fibronectin expression

[0100]

(Fibronectin immunostaining method)

After immobilizing the mouse cornea fragment for 10 minutes at room temperature in 4% paraformaldehyde, 0.5% TritonX-100 was added and allowed to penetrate for 5 minutes. Added 1% bovine serum albumin (BSA), and the fragment was incubated for 1 hour at room temperature. The corneal fragment was then immersed in the primary antibody solution and reacted overnight at 4°C. A 1:250 diluted solution of fibronectin polyclonal antibody (abcam) was used as the primary antibody. Then, the cornea fragment was immersed in a mixture of secondary antibody solution and DAPI solution (Dojindo Laboratories), and they reacted for 1 hour at room temperature away from light. A 1:2000 dilution of Alexa Fluor® 488-labeled goat anti-rabbit IgG (Life Technologies) was used as a secondary antibody. A diluted 1:1000 DAPI solution was used. The corneal fragment was stretched and placed on a glass slide and covered with a coverslip. The prepared sample was observed using an Olympus FV1000 FLUOVIEW confocal laser scanning microscope (Olympus Corporation) or an LSM880 confocal laser scanning microscope (Carl Zeiss Microscopy Co., Ltd.). The central section, corneal regions in the 6 o'clock direction, 9 o'clock direction (ear side) and 3 o'clock direction (nose side) were observed with a 40x or 100x field of view.

[0101]

(Results)

A trend towards suppression of the decrease in corneal endothelial density in the central part of the cornea was observed in the EW-7197 eye drop group up to week 6, but no significant difference in corneal endothelial density in the central part of the cornea was observed between the EW-7197 eye drop group and the eye drop group. drops of the base agent at week 12. Since the droplets tended to be larger in the peripheral cornea than in the central cornea, it is clear that no significant difference in corneal endothelial density was observed in the central cornea between the EW-7197 eye drop group and the base agent eye drop group at week 12 . Indeed, fibronectin expression levels tended to be higher in the peripheral region than in the central region (Figure 9). As shown in Figure 9, a clear suppression of fibronectin expression was observed in the 0.02% and 0.1% EW-7197 eye drop groups. When the same experiment was carried out with a concentration of 0.004% (0.1 mM), which was 1/5 of the concentration of 0.02% EW-7197 eye drops, to test the pharmacological effect at a low concentration, a trend of suppression of fibronectin expression was observed. This showed that EW-7197 is effective over a wide range of concentrations.

[0102]

Example 7: Method for Formulating a Cornea Preservation Solution Containing EW-7197

In this example, a corneal preservation solution containing EW-7197 was prepared by the following method as a formulation example.

[0103]

The preservation solution shown below is prepared in a conventional manner.

[0104]

EW-7197 effective amount (e.g. 0.1 µM)

Optisol-GS (Bausch-Lomb) suitable quantity

Total amount 100 ml

Example 8 Eye Drop Production Example

The composition of the test compound at each concentration is shown below.

[0105]

EW-7197 0.1 mM

Sodium chloride 0.85 g

Sodium dihydrophosphate dihydrate 0.1 g

(optional) benzalkonium chloride 0.005 g

Sodium hydroxide suitable amount

Purified water suitable amount

Total amount: 100 ml (pH 7.0)

The concentration can be diluted using a base agent consisting of the following components.

[0106]

Sodium chloride 0.85 g

Sodium dihydrophosphate dihydrate 0.1 g

(optional) benzalkonium chloride 0.005 g

Sodium hydroxide suitable amount

Purified water suitable amount

Total amount 100 ml (pH 7.0)

For example, commercially available components that comply with the Japanese Pharmacopoeia or its equivalents can be used for each component other than the active ingredient.

[0107]

Example 9: Diagnosis and Therapy Example

The present invention is useful in the diagnosis of Fuchs endothelial corneal dystrophy or a similar corneal endothelial disease (specific examples thereof include 1) observation of droplet formation, thickening of the Descemet's membrane, corneal epithelial edema, or corneal stromal edema by slit microscopy, 2) observation of images of droplets or corneal endothelial disturbance using a specular microscope, 3) observing corneal edema with Pentacam, OCT, ultrasonic corneal thickness gauge and the like, and 4) when determining high risk by genetic diagnosis). It can be treated using the composition of the present invention in the form of eye drops, intracameral injection, administration using a sustained release agent, intravitreal injection, or subconjunctival injection.

[0108]

For example, commercially available components that comply with the Japanese Pharmacopoeia or its equivalents can be used for each component other than the active ingredient.

[0109]

As described above, the present invention is illustrated by the application of its preferred embodiments. However, it is clear that the scope of the present invention should be interpreted solely on the basis of the claims. It is also intended that any patent, any patent application and any references cited in the present invention should be incorporated into the present invention by reference in the same manner as if the contents were specifically described in the present invention. The present application claims priority over Japanese Patent Application No. 2017-239049 (filed December 13, 2017) and Japanese Patent Application No. 2018-184783 (filed September 28, 2018). Their content is incorporated into the present invention by reference.

[Industrial applicability]

[0110]

Provide a drug for the treatment or prevention of a condition, disorder or disease of the corneal endothelium, containing (T)EW-7197. In particular, a medicament is provided for treating or preventing corneal endothelial disorder in Fuchs endothelial corneal dystrophy. Provide technology that is available in industries (pharmaceutical industry and the like) associated with technology related to the development of drugs, or the like, based on this technology.

Claims (10)

1. The use of a compound for the treatment or prevention of a condition, disorder or disease of the corneal endothelium, wherein the compound is (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridine -6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof. 2. Use according to claim. 1, where the compound is intended to suppress the decrease in the density of corneal endothelial cells. 3. Use according to claim 1 or 2, wherein the condition, disorder or disease of the corneal endothelium is Fuchs endothelial corneal dystrophy or endothelial corneal changes in the form of drops. 4. Use according to claim 1, wherein the condition, disorder or disease of the corneal endothelium results from overexpression of the extracellular matrix (ECM). 5. Use according to claim 4, wherein the extracellular matrix (ECM) is selected from the group consisting of type I collagen, type IV collagen, type V collagen and fibronectin. 6. Use according to claim 4 or 5, wherein the condition, disorder, or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, drop formation, Descemet's membrane thickening, corneal thickening, corneal opacity, scar, corneal opacification, corneal macula , walleye, glare, and defocused vision. 7. Application according to any one of paragraphs. 1-6, wherein the condition, disorder, or disease of the corneal endothelium is Fuchs' corneal endothelial dystrophy. 8. Application according to any one of paragraphs. 1-7, wherein (T)EW-7197, or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, is present in the composition at a concentration of from about 0.001 mM to about 10 mM, where "about" refers to a range of ±10% subsequent numeric value. 9. Application according to any one of paragraphs. 1-8, wherein (T)EW-7197, or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, is present in the composition at a concentration of from about 0.01 mM to about 5 mM, where "about" refers to the range of ±10 % of the next numeric value. 10. Application according to any one of paragraphs. 1-9, where the compound is administered in the form of eye drops.

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